Wnt/β-catenin信号通路介导糖基化终未产物对大鼠骨髓间充质干细胞成骨分化的影响

Effect of Wnt/β-catenin signaling pathway induced advanced glycation end products on osteogenic differentiation of rat bone marrow mesenchymal stems cells

目的探讨糖基化终末产物(advanced glycation end products,AGEs)对体外培养SD大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)增生及成骨分化的影响及可能机制.方法采用全骨髓法分离培养4周龄SD大鼠的BMSCs.将BMSCs随机分为成骨诱导液、BSA组、AGEs3组,Western blot法检测AGEs对BMSCs成骨分化过程中β-catenin、OSX、RUNX2、RAGE(AGE受体)蛋白表达量的影响,茜素红染色观察AGEs对BMSCs成骨分化过程中矿化的影响.抑制RAGE后实时荧光定量聚合酶链反应(real-time fluo-rescence quantitative polymerase chain reaction,RT-PCR)及Western blot法再次测定上述指标水平.结果0.2 g/L AGEs作用于体外培养SD大鼠BMSCs,上调RAGE蛋白表达(0.88±0.04),β-catenin、OSX、RUNX2蛋白表达降低(分别为0.21±0.02,0.25±0.03和0.21±0.01,P<0.05).RAGE中和抗体阻断RAGE作用后,AGE+RAGE中和抗体组RAGE蛋白表达下调(0.30±0.03),β-catenin、OSX、RUNX2蛋白表达增高(0.90±0.02,0.84±0.03和0.88±0.02,P<0.05).结论AGEs通过Wnt/β-catenin信号通路抑制BMSCs成骨分化.

Objective To investigate the effects and the possible mechanism of receptor for advanced glycation end products(AGEs)on proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)in SD rats cultured in vitro.Methods BMSCs were isolated and cultured from SD rats aged 4 weeks by whole bone mar-row method.BMSCs were randomly divided into three groups:osteogenic induction fluid,BSA groups and AGREs group,Western blot method was used to detect the effects of AGEs on protein expression ofβ-cateninm,OSX,RUNX2 and RAGE in the process of osteoblastic differentiation of BMSCs,and alibi red staining was used to observe the effect of AGEs on mineralization in the process of osteoblastic differentiation of BMSCs.Real-time fluorescence quantitative poly-merase chain reaction and Western blot assay were used to determine the above indexes again after RAGE suppression.Results 0.2 g/L AGEs acted on BMSCs of SD rats cultured in vitro,upregulation of RAGE protein expression(0.88±0.04),decreased protein expression ofβ-catenin,OSX,and RUNX2(0.21±0.02,0.25±0.03,and 0.21±0.01,P<0.05).After RAGE was blocked by RAGE neutralizing antibodies,RAGE protein expression(0.30±0.03)of AGE+RAGE neutralizing antibody group is down-regulated,β-catenin,OSX,and RUNX2 expression(0.90±0.02,0.84±0.03,and 0.88±0.02)are increased(P<0.05).Conclusion The AGEs goes through theβ-catenin signal pathway to inhibit BMSCs osteogenic differentiation.